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Description: TDG is a thermostable thymine DNA glycosylase from Methanobacterium thermoautotrophicum. The optimal temperature for the enzyme is 65°C. The enzyme lacks significant AP lyase or endonuclease activity. TDG works effectively in heteroduplex analysis to detect C to T transitions.Source: Thermostable TDG is purified from E. coli containing a recombinant plasmid harboring the Methanobacterium thermoautotrophicum TDG gene.Unit Definition: One Unit is the amount of enzyme required to cleave 1 pmole of an oligonucleotide duplex containing a T/G mismatch in 1 hour at 65°C. Only the strand containing the T is cleaved.Substrate Specificity: TDG enzyme recognizes T/G mismatches in duplex DNA and cleaves the strand with the T. The opposite strand is not cleaved. The enzyme also recognizes G/G mismatches if at least one nearest neighbor is an A or T and nicks one strand or the other. The enzyme exhibits poor AP lyase activity.Assay Conditions & Analysis: 4 pmoles of T/G mismatch oligonucleotide set with the T oligo end-labeled, 1X REC™ Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10mM EDTA), and serial dilutions of enzyme in a 20 μl reaction volume are incubated for 1 hour at 65°C. To complete cleavage of abasic site, fresh 1N NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95°C. For analysis, 24 μl of 2X Loading Buffer (20 mM EDTA, 95% formamide, and 0.13% bromophenol blue)are added, and the samples heated at 95°C for 10 min then fast cooled to 4°C. The cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified.Storage Buffer: 25 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol.Storage Conditions: Store at -20°C in a manual defrost freezer. For long term storage, freeze in working aliquots at -80°C. Avoid repeated freeze-thawing. Enzyme may be diluted in 1X REC Buffer 4 for immediate use. TDG protein in storage buffer can survive for up to 24 hours at 37°C with less than 10% loss in activity.